The Par polarity proteins are of profound importance to the morphogenesis of all metazoans, and defects in polarity are associated with cancer. Par3 sits at the apex of a signaling network that controls cell polarity, and we discovered two crucial downstream functions of Par3 in epithelial cells - to deliver atypical protein kinase C (aPKC) to the apical cortex and to regulate the Rac GTPase. Our goal is to determine the outputs to this signaling network, using the mammary gland as an in vivo model, and MDCK 3D cultures in vitro. We developed a powerful method for studying gene function in mouse mammary gland, identified a key role for Par3 in mammary ductal organization, and discovered important functions for Par3 in the control of apoptosis and Rac-dependent proliferation. We also showed that apical aPKC is essential for maintaining correct spindle orientation. In Drosophila Par3 is needed for stem cell function, but loss of Par3 does not cause defects in mouse mammary stem cell function. However, a Par3-related gene exists in vertebrates, which we discovered and named Par3L. We have exciting preliminary data that Par3L plays an unexpected role in the mammary stem cell maintenance. Based on this work we will focus on the following aims: 1. Determine the molecular mechanisms underlying the delivery of aPKC to the apical cortex of epithelial cells. A central, unanswered question is why Par3 is needed for aPKC localization, since in principle the aPKC could diffuse on its own to the apical cortex. We will test the hypothesis that Par3 functions as a chaperone for aPKC, preventing the inappropriate interaction of this kinase with other cytoplasmic proteins. We will also test the hypothesis that a phosphatase, bound to Par3, maintains it a dephosphorylated state, to prevent premature disassociation of aPKC until arrival at the apical domain. 2. Determine the mechanisms by which Par3 restrains proliferation and apoptosis. We discovered that loss of Par3 causes multiple defects in mammary development, including both hyper-proliferation and increased apoptosis. We will test the hypothesis that hyper-proliferation is a cell non-autonomous response to apoptosis driven by Rac->JNK signaling, and determine if the high apoptosis/proliferation that occurs normally in terminal end buds during ductal outgrowth is mediated by reduced Par3 signaling. 3. Determine the biological function of the Par3-related protein, Par3L, in mammary gland development and stem cell maintenance. We will test the hypothesis that Par3L is essential for mammary stem cell maintenance, and that it functions through recruitment of the master kinase and polarity protein, LKB1. We found that Par3L, but not Par3, binds LKB1. Loss of Par3L reduces mammary gland regeneration efficiency. We will employ a transgenic mouse in which multipotent mammary stem cells are specifically tagged with GFP to measure stem cell survival. We will determine if Par3L is required for LKB1 activity in stem cells, and test the function of mutants of Par3L that cannot bind LKB1. PUBLIC HEALTH RELEVANCE: Cell polarity is a fundamental attribute of life. Polarity is essential in multicellular organisms for every aspect of development, and is defective in cancer. All multicellular organisms possess similar molecular machinery that controls cell polarity that has been conserved throughout evolution. This machinery contains the Par polarity proteins. The goal of this proposal is to determine how the Par proteins organize epithelial cells to create a top (apical domain) and a bottom (basal domain), and to control proliferation and cell death. In addition, we believe that Par proteins play a vital role in the survival of adult stem cells and we will test this idea using mouse mammary glands.